BioMicroCenter:Microarrays: Difference between revisions

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[[Image:Presentation7.jpg|right|300px]]
[[Image:Presentation7.jpg|right|300px]]
Microarray technology is the principle service offered by the MIT BioMicro Center. BMC has extensive experience with several different types of samples and platforms. Microarray labeling and hybridization are typically preformed by BioMicro Center technicians, however our scanners, hybridization ovens and wash stations are available by request.  
The MIT BioMicro Center has extensive experience with several different types of microarray samples and platforms. Microarray labeling and hybridization are typically performed by BioMicro Center technicians, however our scanners, hybridization ovens and wash stations are available by request. NOTE: Assisted Agilent Microarray serves are no longer available. The Agilent oven and microarray scanner is available to users who prepare their own arrays.


== BioMicro Center Services ==
== BioMicro Center Services ==
We are currently set up to primarily handle expression analysis. Our service includes:
We are currently set up to primarily handle expression analysis. Our service includes:


* Evaluation of sample quality using the [[BioMicroCenter:2100BioAnalyzer|Agilent BioAnalyzer 2100]]
* Evaluation of sample quality using the [[BioMicroCenter:Advanced_analytical_Fragment_analyzer|Fragment Analyzer]]
* Sample labeling and clean up.
* Sample labeling and clean up
* Array Hybridization.
* Array Hybridization
* Data extraction and normalization.
* Data extraction and normalization
 
We also have discounts with different vendors and can help you purchase your arrays.
We also have discounts with different vendors and can help you purchase your arrays.
<br>
<br>
Line 22: Line 21:
  ! Min.RNA
  ! Min.RNA
  ! Min.Vol.
  ! Min.Vol.
! Platforms
  ! Notes
  ! Notes
  |-
  |-
Line 27: Line 27:
  | 1 μg
  | 1 μg
  | 6 μL
  | 6 μL
  | labeling using [http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion] for Affy arrays
| Affymetrix
  | labeling using [http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion] for Affymetrix arrays
  |-
  |-
  | 3' Array (std)
  | 3' Array (std)
  | 100 ng
  | 50ng / ''100pg ''
  | 6 μL
  | 6 μL
  | labeling using NuGEN for Affy arrays.
| Affymetrix
  | labeling using Affymetrix reagents
  |-  
  |-  
  | Exon Array
  | Exon Array
  | 100 ng
  | 50ng / ''100pg''
  | 6 μL
  | 6 μL
  | labeling using NuGEN for Affy arrays
  | Affymetrix
  |-
  | labeling using Affymetrix reagents
| small RNA
| N/A
| N/A
|BMC recommends the use of multiplexed [[BioMicroCenter:Sequencing|Illumina sequencing]] for small RNA samples
|}
|}
<br>
<br>
=== SAMPLE SUBMISSION ===
=== SAMPLE SUBMISSION ===


We encourage researchers to contact us several days in advance of sample submission to allow us lead time to purchase any specialized Affymetrix or Agilent arrays. While we keep a few arrays in stock, most arrays are ordered on an as needed basis. A list of some of the available arrays and their prices can be found [[Media:Affy_Arrays_081218.pdf| in this file]]. Information about Affymetrix arrays is available at http://www.affymetrix.com/products_services/index.affx
We encourage researchers to contact us several days in advance of sample submission to allow us lead time to purchase any specialized arrays. All arrays are ordered on an as needed basis. A list of some of the available arrays and their prices can be found [[Media:Affy_Arrays_081218.pdf| in this file]]. Information about Affymetrix arrays is available at http://www.affymetrix.com/products_services/index.affx


All submissions must be accompanied by a [[BioMicroCenter:Forms#Downloads | Affymetrix Submission Form]].  
All submissions must be accompanied by a [[BioMicroCenter:Forms#Downloads | Affymetrix Submission Form]].  
<br>
<br>
=== TURN AROUND ===
=== TURN AROUND ===
The BioMicro Center will turn around your samples as quickly as possible. We are currently exploring methods of increasing our sample throughput to reduce turn around time. Currently the average turn around time is currently 2-3 weeks.
The BioMicro Center will turn around your samples as quickly as possible. Currently the average turn around time is 1-2 weeks.
<br>
<br>


=== NON-MIT USERS ===
== ARRAY SELECTION ==
Please contact [[BioMicroCenter:People|Manlin Luo]] to arrange a drop off date and time. Samples on dry ice and chips at room temperature with a completed order form are shipped by overnight delivery to:
Biomicro Center staff are available to assist you in selection of the proper microarray design. Newer Affymetrix microarrays take in to account ENCODE and RNAseq data to produce more efficient designs while older arrays have many probesets that are less useful. BioMicro Center staff can also assist you in selecting Agilent arrays but we are not able to assist in processing those microarrays.
  MIT BioMicro Center
  Attention: Manlin Luo
  31 Ames Street, Building 68-316
  Cambridge, MA 02139
<br>
=== [[BioMicroCenter:Prices|PRICES]] ===
Please note that processing prices '''do not''' include the cost of the array. In order to be sure we have arrays on hand, we encourage users to contact us several days in advance of submitting samples.


{| border=1  style="text-align:center"
Please note that the BioMicro Center no longer supports printing of microarrays in house. Custom oligonuclotide arrays are available from Agilent at the same cost as their commercial arrays using their [http://www.chem.agilent.com/en-US/products/instruments/dnamicroarrays/pages/gp50660.aspx eArray system] (we can [[BioMicroCenter:Computing#BioInformatics_Services|help you]] through the process of designing arrays). For anyone requiring microarray printing, we encourage you to contact [http://jura.wi.mit.edu/genomecorewiki/index.php/Tom_Volkert Tom Volkert at the Whitehead Institute CGT].
! ITEM
! style="width:100px"|[[CoreDeps|CORE LAB]]  
! style="width:100px"| MIT
! style="width:100px"| NON-MIT
! style="width:100px"| unit
|-
| Affy 3' Processing || $260 || $430 || $645 || per array
|- 
| Affy Exon Processing || $300 || $535 || $800 || per array
|-
| Affy Prokaryote || $260 || $430 || $645 || per array
|-
| Agilent Arrays || (inquire) || || ||
|}


Common Affymetrix Arrays (some in stock):
== QUALITY CONTROL ==
* Human U133 Plus 2.0 / Mouse 430 2.0 - '''$400'''
The BioMicro Center employs several quality controls as part of microarray analysis
* Human U133A 2.0 / Mouse 430A 2.0 - '''$250'''
=== RNA ===
* Human / Mouse Exon 1.0 ST - '''$400'''
All RNA samples are run on the [[BioMicroCenter:Advanced_analytical_Fragment_analyzer|Fragment Analyzer]] prior to labeling. The Fragment Analyzer results are used to determine the concentration of the RNA and the quality of the RNA based on their [[BioMicroCenter:RIN|RNA Integrity Number (RIN)]] score.
* Yeast 2.0 - '''$175'''
Full Price List: [[Media:Affy_Arrays_081218.pdf| Affymetrix Prices]]


** Please note: Processing prices will be changing on July 1, 2009 **
=== Affymetrix Arrays ===
After scanning, 3' Affymetrix arrays are processed through a number of quality control steps to confirm the quality of the array. These steps create quality control plots that we distribute to the user.


== Platforms ==
==== QC PLOTS ====
In all cases, the plots are guidelines but not absolutes. Differences in the quality of starting RNA or source of the RNA can have a major impact on the plots. Biological replicates should behave similarly and we focus on changes where we find that is not true.


The BioMicro Center currently processes two types of Arrays:
The RNA Degradation plot measure the relative intensity of probes at the 5' and 3' end of the average gene. Differences in the reverse transcription step can hive significant effects on the outcome. The precise slope of the line is not as important as having the lines have similar slopes.


* [http://www.affymetrix.com AFFYMETRIX] - Affymetrix offers one color arrays for a variety of assays including expression, ChIP-chip, and SNP analysis. We routinely use Affymtrix arrays for the quantification of 3' ends and whole transcripts (exon arrays) from both eukaryotes and prokaryotes. The BioMicro Center has a hybridization oven, two 450 fluidics devices and a 7G scanner for handling Affymetrix arrays. As part of our effort to control costs, the BioMicro Center is part of the Core Lab program, which lowers our annual maintenance contract and allows us to maintain a number of standard array types in stock.  
Normalized Unscaled Standard Error (NUSE), provides a measure of relative chip quality derived from the residuals from the RMA model. NUSE scores above 1.05 or disconnected from the others are generally (though not always) considered poor.


* [http://www.agilent.com AGILENT] - Agilent offers two-color arrays for a variety of assays including gene expression, ChIP-chip, miRNA hybridization, and CGH. The MIT BioMicro Center hosts a hybridization oven and a high-throughput Agilent Microarray Scanner donated by the Bell lab. We do not currently maintain a stock of Agilent arrays but do have a significant discount on the purchase of arrays.  
The Affy QC pdf contains a number of metrics including box plots and histograms of intensity, QC scores for spike in controls and interarray correlation.  


Please note that the BioMicro Center no longer supports self printed arrays. Custom oligonuclotide arrays are available from Agilent at the same cost as their commercial arrays using their [http://www.chem.agilent.com/en-US/products/instruments/dnamicroarrays/pages/gp50660.aspx eArray system] (we can [[BioMicroCenter:Computing#BioInformatics_Services|help you]] through the process of designing arrays). For anyone requiring self printed arrays, we encourage you to contact [http://jura.wi.mit.edu/genomecorewiki/index.php/Tom_Volkert Tom Volkert at the Whitehead Institute CGT].
Fianlly, we use gcRMA for normalization. gcRMA normalized data is included in the data we distribute.


== Technology Updates ==
==== References ====
These plots are generated using a number of packages from [http://www.bioconductor.org/ bioconductor] including:


=== Labeling Kits ===
*affy:Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315.
[[Image:BMC_NugenKit.png|right]]
*affyQCReport
Affymetrix is retiring their old 3' Array kits in September 2009. In response, BMC has been evaluating new technologies. The newer kits are faster and require less starting material. We have selected the NuGEN technology as the primary labeling method we will be using going forward for eukaryotic arrays because of their ease and the modularity of their system that allows us to combine the processing of Affymetrix 3', Affymetrix Exon, and Agilent arrays. Information on the evaluation can be found '''[[BioMicroCenter:3primeIVT|HERE]].'''. We DO NOT RECOMMEND mixing labeling types within an experiment as we have observed large differences between the absolute intensities of signals between labeling methods. Arrays can still be hybridized using the Affymetrix 3'IVT, Affymetrix Exon and old Affymetrix 3' array by request (please ask for pricing).  
*affyPLM: Brettschneider J, Collin F, Bolstad BM, and Speed TP. (2007) Quality assessment for short oligonucleotide arrays. Technometrics.
*LPE:   Nitin Jain, Michael O'Connell and Jae K. Lee. Includes R source code contributed by HyungJun Cho <hcho@virginia.edu> (). LPE: Methods for analyzing microarray data using Local Pooled Error (LPE) method. R package version 1.20.0. http://www.r-project.org,


We have also switched our prokaryotic labeling system from the Affymetrix system to one developed by [http://www.ambion.com/catalog/ProdGrp.html?fkProdGrp=339 Ambion]. The Ambion system requires significantly less input RNA and has a more streamlined protocol. The Affymetrix labeling system is still available by request.


=== Small Cell Numbers ===
Bioconductor reference:  Bioconductor: Open software development for computational biology and bioinformatics R. Gentleman, V. J. Carey, D. M. Bates, B.Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, and others 2004, Genome Biology, Vol. 5, R80


NuGEN has recently developed kits that allow the analysis of very [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-pico/ small amounts of purified RNA (100pg)] or even [http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/wt-ovation-one-direct/ single cells]. Please contact [[BioMicroCenter:People|Stuart Levine]] if you are interested in trying out these technologies.
gcRMA:   Jean(ZHIJIN) Wu and Rafael Irizarry with contributions from James MacDonald Jeff Gentry (). gcrma: Background Adjustment Using Sequence Information. R package version 2.18.1.


== Analysis and Data Retrieval ==
== DATA ANALYSIS ==


=== Affymetrix Data Analysis ===
=== Affymetrix Data Analysis ===


BioMicro Center will perform the initial data extraction of your samples using Affymetrix GCOS V.1.4. Affymetrix data is scaled to a average intensity of 500 per chip. Six files for each sample will be provided to the user:
BioMicro Center uses Affymetrix Expression Console and GCRMA for analysis of Affymetrix data. Several files for each sample will be provided to the user:


* .exp: Experiment information file  
* .exp: Experiment information file  
Line 129: Line 107:


=== Agilent Data Analysis ===
=== Agilent Data Analysis ===
Agilent Scanner is available to members of the MIT Community without charge. If you are the first time user, contact [[BioMicroCenter:People|Stephen Goldman]] to set up a bionet account for your data storage. For questions about equipment, contact [[BioMicroCenter:People|Manlin Luo]]
Agilent Scanner is available to members of the MIT Community. We are currently running Feature extractor version 10.5. Please use [https://mit.ilabsolutions.com/sc/3381/ki-genomics-core-mit-biomicro-center/?tab=equipment iLabs] to book time on the instrument.  
 
After you have completed your scan, please make sure to sign the scanner record form, which is  near the equipment and to remove your files from the computer. CD-Rs are available if needed.
 
 
=== Data Transfer ===
 
You will be notified by email that your data is ready. At that time please pick up any leftover samples and arrays belonging to you. Samples will be held at the facility at -80C for 60 days after your data is generated.
 
The data will be placed on our server and can be downloaded by secure FTP or SSH.
SSH can be downloaded from: ftp://metalab.unc.edu/pub/packages/security/ssh/SSHSecureShellClient-3.2.9.exe
Your user name and password will be included in the email. To obtain your data, enter the following in your client:
 
  Host:      bmc-150.mit.edu
  user:      provided in email     
  Password:  provided in email


Please contact [[BioMicroCenter:People|Stephen Goldman]] if you have difficulty obtaining your data.
== LINKS ==
=== [[BioMicroCenter:Pricing|PRICES]] ===
=== [[BioMicroCenter:FAQ#NON_MIT_USERS|Information for non-MIT labs]] ===
=== [[BioMicroCenter:FAQ#HOW_DO_I_GET_MY_DATA|Obtaining your results]] ===
You will be notified by email that your data is ready. At that time please pick up any leftover samples and arrays belonging to you. Samples will be held at the facility at -80C for 60 days after your data is generated. Information on downloading your data is in the link above.

Revision as of 22:46, 4 June 2016

HOME -- SEQUENCING -- LIBRARY PREP -- HIGH-THROUGHPUT -- COMPUTING -- OTHER TECHNOLOGY

The MIT BioMicro Center has extensive experience with several different types of microarray samples and platforms. Microarray labeling and hybridization are typically performed by BioMicro Center technicians, however our scanners, hybridization ovens and wash stations are available by request. NOTE: Assisted Agilent Microarray serves are no longer available. The Agilent oven and microarray scanner is available to users who prepare their own arrays.

BioMicro Center Services

We are currently set up to primarily handle expression analysis. Our service includes:

  • Evaluation of sample quality using the Fragment Analyzer
  • Sample labeling and clean up
  • Array Hybridization
  • Data extraction and normalization

We also have discounts with different vendors and can help you purchase your arrays.

SAMPLE TYPES

The BioMicro Center has extensive experience handling samples for expression analysis.

Eukaryote Min.RNA Min.Vol. Platforms Notes
Prokaryote 1 μg 6 μL Affymetrix labeling using Ambion for Affymetrix arrays
3' Array (std) 50ng / 100pg 6 μL Affymetrix labeling using Affymetrix reagents
Exon Array 50ng / 100pg 6 μL Affymetrix labeling using Affymetrix reagents


SAMPLE SUBMISSION

We encourage researchers to contact us several days in advance of sample submission to allow us lead time to purchase any specialized arrays. All arrays are ordered on an as needed basis. A list of some of the available arrays and their prices can be found in this file. Information about Affymetrix arrays is available at http://www.affymetrix.com/products_services/index.affx

All submissions must be accompanied by a Affymetrix Submission Form.

TURN AROUND

The BioMicro Center will turn around your samples as quickly as possible. Currently the average turn around time is 1-2 weeks.

ARRAY SELECTION

Biomicro Center staff are available to assist you in selection of the proper microarray design. Newer Affymetrix microarrays take in to account ENCODE and RNAseq data to produce more efficient designs while older arrays have many probesets that are less useful. BioMicro Center staff can also assist you in selecting Agilent arrays but we are not able to assist in processing those microarrays.

Please note that the BioMicro Center no longer supports printing of microarrays in house. Custom oligonuclotide arrays are available from Agilent at the same cost as their commercial arrays using their eArray system (we can help you through the process of designing arrays). For anyone requiring microarray printing, we encourage you to contact Tom Volkert at the Whitehead Institute CGT.

QUALITY CONTROL

The BioMicro Center employs several quality controls as part of microarray analysis

RNA

All RNA samples are run on the Fragment Analyzer prior to labeling. The Fragment Analyzer results are used to determine the concentration of the RNA and the quality of the RNA based on their RNA Integrity Number (RIN) score.

Affymetrix Arrays

After scanning, 3' Affymetrix arrays are processed through a number of quality control steps to confirm the quality of the array. These steps create quality control plots that we distribute to the user.

QC PLOTS

In all cases, the plots are guidelines but not absolutes. Differences in the quality of starting RNA or source of the RNA can have a major impact on the plots. Biological replicates should behave similarly and we focus on changes where we find that is not true.

The RNA Degradation plot measure the relative intensity of probes at the 5' and 3' end of the average gene. Differences in the reverse transcription step can hive significant effects on the outcome. The precise slope of the line is not as important as having the lines have similar slopes.

Normalized Unscaled Standard Error (NUSE), provides a measure of relative chip quality derived from the residuals from the RMA model. NUSE scores above 1.05 or disconnected from the others are generally (though not always) considered poor.

The Affy QC pdf contains a number of metrics including box plots and histograms of intensity, QC scores for spike in controls and interarray correlation.

Fianlly, we use gcRMA for normalization. gcRMA normalized data is included in the data we distribute.

References

These plots are generated using a number of packages from bioconductor including:

  • affy:Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315.
  • affyQCReport
  • affyPLM: Brettschneider J, Collin F, Bolstad BM, and Speed TP. (2007) Quality assessment for short oligonucleotide arrays. Technometrics.
  • LPE: Nitin Jain, Michael O'Connell and Jae K. Lee. Includes R source code contributed by HyungJun Cho <hcho@virginia.edu> (). LPE: Methods for analyzing microarray data using Local Pooled Error (LPE) method. R package version 1.20.0. http://www.r-project.org,


Bioconductor reference: Bioconductor: Open software development for computational biology and bioinformatics R. Gentleman, V. J. Carey, D. M. Bates, B.Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, and others 2004, Genome Biology, Vol. 5, R80

gcRMA: Jean(ZHIJIN) Wu and Rafael Irizarry with contributions from James MacDonald Jeff Gentry (). gcrma: Background Adjustment Using Sequence Information. R package version 2.18.1.

DATA ANALYSIS

Affymetrix Data Analysis

BioMicro Center uses Affymetrix Expression Console and GCRMA for analysis of Affymetrix data. Several files for each sample will be provided to the user:

  • .exp: Experiment information file
  • .dat: The image of the scanned probe array.
  • .cel: Cell Intensity File derived from .dat file.
  • .chp: Output file generated from the analysis of a probe array.
  • .rpt: Report File generated.
  • .txt: Text File is the same information as the .chp file in text format.

Additional data analysis help is available on a case by case basis. Please contact Stuart Levine if you need additional help with your analysis.

Agilent Data Analysis

Agilent Scanner is available to members of the MIT Community. We are currently running Feature extractor version 10.5. Please use iLabs to book time on the instrument.

LINKS

PRICES

Information for non-MIT labs

Obtaining your results

You will be notified by email that your data is ready. At that time please pick up any leftover samples and arrays belonging to you. Samples will be held at the facility at -80C for 60 days after your data is generated. Information on downloading your data is in the link above.

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